The present invention relates to recombinant conglutinin having anti-virus activities (neutralization activities) which are expected to be applied to medicines and producing method thereof, and a method for detecting physiological activities of collecting.
Conglutinin is an animal lectin belonged to calcium-dependent mammalian C-type lectin family and existed in the bovine serum. Whole amino acids sequence (SEQ ID No.: 1) had been analyzed by Lee et al., [Lee et al., J Biol. Chem., Vol. 266, pp. 2715-2723, 1991].
C-type lectin comprises basic unit having the four unique regions of (1) N-terminal region contained much cysteine, (2) collagen-like region, (3) neck region and (4) carbohydrate recognition domain (CRD) [Malhortra et al., European Journal Immunology, Vol. 22, pp. 1437-1445, 1992].
Besides conglutinin, C-type lectin includes Mannan-Binding Proteins (MBP), Surfactant Protein A (SP-A) and Surfactant Protein D (SP-D), and they are generally called as collectin.
In vertebrates, mechanisms involving specific antibody reaction and immune response through the cells are considered as a main host-defense system against inversion of the pathogenic bacteria. However, recently, non-specific immune response by these lectins seems that it may play an important role to neutralize and remove the various microorganisms in the puerile subjects having the maternal transmigration antibody and the undeveloped specific defense system [Super et al., Lancet, Vol.II, pp. 1236-1239, 1989].
Regarding the role of these lectins on biological defense in host organism, it is reported that infection will be easily spread by, for example, the reduction of the mannan-binding protein concentration in blood due to genetic mutation of the mannan-binding protein [Sumiya et al., Lancet, Vol. 337, pp. 1569-1570, 1991].
The present inventor once reported that the conglutinin and the mannan-binding protein inhibit infection and hemagglutination inhibition activity of H1 and H3 Type Influenza A Viruses (Wakamiya et al., Glycoconjugate J., Vol. 8, p. 235, 1991; Wakamiya et al., Biochem. Biophys. Res. Comm., Vol. 187, pp. 1270-1278, 1992).
Thereafter, the research group of the present inventor isolated cDNA clone encoding the conglutinin and found that there is the closer correlation between gene of the conglutinin and that from the various surfactant protein-D [Suzuki et al., Biochem. Biophys. Res. Comm., Vol. 191, pp. 335-342, 1993].
Accordingly, the conglutinin have been expected as useful material for physiologically active medicine component, but amount of the conglutinin to be obtained from the bovine serum is less. Further, continuous production of the conglutinin is quite difficult because source thereof is completely depended on an animal body. Expression of the conglutinin in Escherichia coli by the genetic recombinant techniques had been tried to realize the large scale production of the conglutinin.
In such process, first of all, whole cDNA of the conglutinin was amplified by PCR (Polymerase Chain Reaction) method, then the amplified genes were introduced into the expression vector pRSET-A and were expressed with M13/T7 phage. The recombinant conglutinin obtained was analyzed. Although expression of the recombinant conglutinin had been confirmed, expressed amounts are less to be barely detected by Western blotting. This approach is inconvenient to the large-scale production of the conglutinin.
Similar methods had also been tried by using another expression vectors, but the same or less expression level had merely detected by any of the vectors. Anyway, an effective expression system have not yet been realized in the art. This seems due to difficulties in expressing the conglutinin because Escherichia coli does not possess proteins of the structure like collagen-like region. Further, yield of the conglutinin produced from an eukaryotic cells is little, and some of the conglutinin may sometimes have an inappropriate post-transcriptional modification.
As stated above, although the conglutinin have been expected as an useful medicine component, neither the natural source nor the genetic recombinant techniques could provide the large amount of the conglutinin.
The present inventions are established to solve the aforenoted problems in the prior art, and they are based on the findings that large amount of the present recombinant conglutinin can be produced according to the previously noted expression system, wherein the recombinant conglutinin comprises (i) a part of the collagen region of the conglutinin consisting of 171 amino acids sequence (SEQ ID No.: 2), namely, an extremely short collagen region consisting of six amino acids comprising two units of amino acids sequence of Gly-Xaa-Xaa (SEQ ID No.: 3; 2nd and 3rd amino acids are protein-constituting amino acid), (ii) the neck region and (iii) the carbohydrate recognition domain.
Despite that the recombinant conglutinin of the present invention comprises the extremely short collagen region, the neck region and the carbohydrate recognition domain, they maintain the similar activities to be expressed by the native conglutinin including the activities of the sugar binding specificities, conglutination activities depending on calcium, hemagglutination inhibition (HI) activities against Influenza A viruses, neutralization activities and viral growth (infection spread) inhibition activities.
Further, the method for detecting the physiological activities of the collectins can be used to detect the physiological activities of the collectins including the conglutinin by evaluating inhibition effects on budding of the viruses from the cells preinfected with viruses, in particular, with influenza A virus. According to this method, physiological activities of the collectins can exactly be detected even if they were once determined as inactive by the conventional detection method (e.g., detection by neutralization activities), physiological activities of the collectins would therefore be appropriately evaluated from different aspects. Further, the present detection method may provide a landmark to determine a preferable use of the collecting.